Effect of Liposome Encapsulated Vesicular Stomatitis Virus Matrix Protein in Hematological Malignancy

Objective: Vesicular Stomatitis Virus (VSV) has shown antitumor effects in many studies, however as an active virus,it has biosafety problems when being used in human body. Matrix protein (M protein) of VSV also has antitumor effects without the existence of other portions of VSV.Recently liposome is widely used as a gene delivery vector. So the present study was to investigate the effect of liposome as a gene delivery vector in hematological malignant cell lines and to investigate the effect of liposome encapsulated Vesicular Stomatitis Virus Matrix protein in hematological malignancy in vitro.

Methods: First, Lipofectamine 2000 was selected as a kind of gene delivery vector and its efficacy in different cell lines was identified by transfecting the plasmid DNA encoding GFP gene into suspension cell lines, including K562 cells (human chronic myeloid leukemia), Jurkat cells (human T cell lymphoma), Raji cells (human B cell lymphoma) and both attachment and suspension cell line CZ-1 cells (human multiple myeloma) according to the instruction provided by the Invitrogen company when the ratios of DNA to Lipofectamine2000 were 1:2,1:2.5,1:3,1:4 and 1:5, respectively. Selecting those cell lines was based on that the transfection efficiency of liposome was above 20% and the ratios which could help to receive the greatest efficiency. The human ovarian cancer cell line A2780 was the positive control and the ratio of DNA to Lipofectamine varied from 1:2.5 to 1:3.

Secondly, in vitro study, the P-M group was the one in which the plasmid DNA encoding M protein of Vesicular Stomatitis Virus (VSV-M) was transfected into the selected cell lines by the best ratios of DNA to Lipofectamine2000.The groups of Lipofectamine2000- treated (LIPO) , Lipofectamine2000 encapsulated empty plasmid pVAX -treated (P) and untreated one (BLK) were set as the control groups. Morphological changes were observed and the cell growth inhibition rates in different groups were measured by MTT (Methylthiazolyldiphenyl-tetrazolium bromide) method at 24h, 48h and 72h after transfection. The cell apoptosis rates of K562 cells were tested by Flow Cytometry at 48h after transfection.

Results

1. The Lipofectamine2000 transfection efficiency in K562 cells could reach up to 50%, while in different conditions the efficiency in CZ-1 was only 20% and that in Raji and jurkat cells was below 10%. The efficiency could also reach 50% in A2780 cells when the ratio was1:3.

2. Study of the effect of liposome encapsulated Vesicular Stomatitis Virus Matrix protein in hematological malignancy: 2.1 K562 and CZ-1 cells: The morphology of K562 and CZ-1 cells in P-M groups was nearly not changed at 24h,48h and 72h after transfection. Futhermore, MTT method showed that, compared with the P and LIPO groups, there were no growth inhibition in K562 and CZ-1 cells at different time points(P>0.05). 2.2 A2780 cells: In the P-M groups, the morphology of A2780 cells was changed ,and a lot of suspension cells were observed in the medium after 48h. Compared with the P and LIPO groups, there was obvious growth inhibition in A2780 cells at different time points (P<0.05).In the P groups, the morphology of A2780 cells was also changed little. Compared with the LIPO group, there was some growth inhibition in A2780 cells at different time points (P<0.05). In the LIPO and BLK groups, these cells had no morphological changes and no growth inhibition.

3. Flow Cytometry showed that there was no difference between the P-M group and P,LIPO groups in K562 cell line in terms of cell apoptosis rates after 48 hours of transfection with VSV-M by means of- liposome(P>0.05).

Conclusions

1. Liposome was not a good choice for the gene transfection in hematological cell lines, including Raji,CZ-1 and Jurkat; however it had good efficacy in K562 and A2780 cell lines.

2. M protein of Vesicular Stomatitis Virus had no obvious inhibition effect in several hematological malignancy cell lines in the current transfection conditions in vitro, while it had good inhibition effect in A2780 cell line. Futhermore, M protein could not induce cell apoptosis in K562 cells in this study.